RESUMO
Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.
Assuntos
Anemia de Fanconi , Neoplasias , Humanos , Sobrevivência Celular/genética , Cromatina/genética , Ciclina E/genética , Ciclina E/metabolismo , Dano ao DNA , Reparo do DNA , Replicação do DNA/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Neoplasias/genéticaRESUMO
Inhibition of WEE1 kinase by AZD1775 has shown promising results in clinical cancer trials, but markers predicting AZD1775 response are lacking. Here we analysed AZD1775 response in a panel of human breast cancer (BC) cell lines by global proteome/transcriptome profiling and identified two groups of basal-like BC (BLBCs): 'PTEN low' BLBCs were highly sensitive to AZD1775 and failed to recover following removal of AZD1775, while 'PTEN high' BLBCs recovered. AZD1775 induced phosphorylation of DNA-PK, protecting cells from replication-associated DNA damage and promoting cellular recovery. Deletion of DNA-PK or PTEN, or inhibition of DNA-PK sensitized recovering BLBCs to AZD1775 by abrogating replication arrest, allowing replication despite DNA damage. This was linked to reduced CHK1 activation, increased cyclin E levels and apoptosis. In conclusion, we identified PTEN and DNA-PK as essential regulators of replication checkpoint arrest in response to AZD1775 and defined PTEN as a promising biomarker for efficient WEE1 cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Proteína Quinase Ativada por DNA/genética , PTEN Fosfo-Hidrolase/genética , Proteínas Tirosina Quinases/genética , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteína Quinase Ativada por DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Tirosina Quinases/metabolismo , ProteomaRESUMO
SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW7α Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation-resistant SOX9 mutant reveals activation of pro-metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Meduloblastoma/metabolismo , Fatores de Transcrição SOX9/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Compostos de Anilina/farmacologia , Animais , Benzamidas , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular , Cromonas/farmacologia , Cisplatino/farmacologia , Proteínas F-Box/genética , Proteína 7 com Repetições F-Box-WD , Quinase 3 da Glicogênio Sintase/metabolismo , Células HEK293 , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Camundongos Nus , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Pirróis/farmacologia , Fatores de Transcrição SOX9/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The mitotic spindle assembly checkpoint (SAC) maintains genome stability and marks an important target for antineoplastic therapies. However, it has remained unclear how cells execute cell fate decisions under conditions of SAC-induced mitotic arrest. Here, we identify USP9X as the mitotic deubiquitinase of the X-linked inhibitor of apoptosis protein (XIAP) and demonstrate that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons. We find that primary human aggressive B-cell lymphoma samples exhibit high USP9X expression that correlate with XIAP overexpression. We show that high USP9X/XIAP expression is associated with shorter event-free survival in patients treated with spindle poison-containing chemotherapy. Accordingly, aggressive B-cell lymphoma lines with USP9X and associated XIAP overexpression exhibit increased chemoresistance, reversed by specific inhibition of either USP9X or XIAP. Moreover, knockdown of USP9X or XIAP significantly delays lymphoma development and increases sensitivity to spindle poisons in a murine Eµ-Myc lymphoma model. Together, we specify the USP9X-XIAP axis as a regulator of the mitotic cell fate decision and propose that USP9X and XIAP are potential prognostic biomarkers and therapeutic targets in aggressive B-cell lymphoma.